The proposed research is a response to the National Institute of Aging Program Announcement PAR-02-049, addressing the mechanisms underlying age-related disease changes in sensory and motor processing (problem #11). Parkinson's Disease is a neurodegenerative disorder that affects over 1 million Americans over the age of 50. Insights into the biochemical basis for idiopathic Parkinson's Disease can be gained by studying pathways impaired in genetic forms of the disease. The ubiquitin pathway has long been linked to Parkinson's Disease, because the aggregated inclusions founding patients, called Lewy Bodies, contain ubiquitin. The three recently identified genes mutated in familial Parkinson's Disease begin to provide direct molecular links to the ubiquitin pathway. The PARK5 gene is thought to encode UCH-L1, a ubiquitin C terminal hydrolase. The PARK1 gene encodes alpha-synuclein, which is found along with ubiquitin in Lewy Bodies. The PARK2 gene encodes parkin, which is an E3 ubiquitin ligase. Parkin belongs to the growing family of RING-IBR-RING ubiquitin ligases, which also includes the Dorfin protein that plays a role in another neurodegenerative disorder, amyotrophic lateral sclerosis. Despite the biological importance of RING-IBR-RING ubiquitin ligases in neurodegenerative disorders, little is known about their mechanism. This proposal addresses the structure and function of the parkin ubiquitin ligase. Parkin is found in complex with some alpha-synuclein, suggesting that alpha-synuclein is part of a larger parkin-based ubiquitin ligase complex. We will use our expertise in structural biology of E3 ubiquitin ligases to understand the molecular function of parkin, and its interactions with other proteins including alpha-synuclein, by addressing the following questions. 1) Which of parkin's known interacting proteins, including alpha-synuclein, binds to parkin directly and forms a stoichiometric complex with parkin in vitro. 2) What are the structural domains in parkin, and which ones are involved in parkin's protein-protein interactions? 3) What is the three dimensionalstructure of the RING-IBR-RING E3 ubiquitin ligase motif in parkin?